Acetylcholine-induced membrane currents and excitatory postsynaptic currents (EPSCs) were recorded from the neurons of rat superior cervical ganglion (SCG) using the whole-cell patch clamp and the two-electrode voltage clamp techniques, correspondingly. The EPSC decay was bi-exponential, with fast and slow components characterized by time constants 5.5 +/- 0.5 msec and 20.4 +/- 1.2 msec (mean +/- SEM; n = 23), respectively. Blocking of these currents by a series of newly synthesized bis-cationic ammonium compounds, the pentamethonium and pentaethonium derivatives, was analyzed. Blocking effects were due to a block of nicotinic acetylcholine receptor (nAChR) open channel, with mean blocker binding rate constants for the fast component three to five times higher than those for the slow component. Dimensions of a nAChR ionic channel were deduced from a relationship between blocking activity of the compounds and the size of the projections of their three-dimensional molecular models on the neuronal membrane plane. The results suggest that there are two populations of nAChRs in rat SCG neurons; while these polulations differ in the rate constants of the binding by the blocker to their open channels, they exhibit similar channel diameter, 11.8 A, at the level at which the blockers bind to the channel.

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Under intracellular recording, we studied the effect of ATP on nerve cells of the rat intact nodose ganglion. The resting membrane potential of the examined neurons was, on average, -60.3 ± 1.4 mV (n = 84); among such units, 88% were classified as C cells. Local application of 2 mM ATP to the surface of the ganglion using a modified laminar flow system led to depolarization of neurons by 7.1 ± 0.9 mV, on average (n = 19). A blocker of P2X receptors, PPADS (100 µM), suppressed these depolarization responses, decreasing their amplitude, on average, to 16 ± 3% (n = 3) of the initial value. The obtained data indicate that an overwhelming majority of neurons of the intact nodose ganglion possess functional P2X receptors on their membranes. The absence of the corresponding responses in a considerable part of neurons of intact spinal ganglia [13][14][15] was, apparently, determined by the fact that Р2Х receptors in the course of the described experiments had enough time to desensitize before ATP reached the effective concentration.

Decay kinetics of the po~tsynaptic excitatory currents (EPSC), distribution of the antibodies specific to different ~z-subunits of neuronal nicotinic acetylcholine receptors (nAChR), and the effects of these antibodies on ACh-induced membrane currents were studied in neurons of different autonomic ganglia of rats. It was shown that a3-, aS-, and aT-subunits were pre.sent in all studied cultured neurons of the rat superior cervical ganglion (SCG), while the a4-subunit was present only in about half of the neurons; this a-subunit distribution differed from that in cultured tntracardial neurons of rats. Two nAChR populations were found in rat SCG neurons, and a series of nAChR populations.were found in routine superior mesenteric ganglion neurons; they differed in kinetics of their ion channel activity, voltage dependence, and the rate of their open channel blockade. The possible functional role of neuronal rtAChR heterogeneity is discussed.Nicotinic acetylcholine receptors (nAChR) belong to an ionotropic membrane receptor family; they are found both in skeletal muscle fibers and neurons. In their molecular structure, neuronal nAChl~ are close to the GABAA, glycine, and serotonin (5-HT 3) receptors. These receptors are formed by five homologous subunits oriented around the central ion channel [1 ]. In contrast to the muscle nAChR, which are very similar in all muscles and in all vertebrate species, neuronal nAChR are greatly variable. At present, eight variants of a-subunits (the ACh-binding subunits) and three variants of fl-subunits are known. Yet, stoichiometry of these subunits in the neuronal rlAChR of each subtype is far from being finally understood.Recently, a few attempts have been made to identify nAChR subtypes expressed in sympathetic ganglion neurons. In particular, the peptides similar in their amino acid sequence to the (a180-~19I) domain of a3-, a4-, a5-, and a7-subunits, which are thought to be expressed in the sympathetic ganglia [2-4 ], were synthesized. This domain was chosen because its amino sequence in each subunit differs from those in all other subunits mentioned above [5 ], and, on the other hand, this domain is located near a tentative ACh binding site. 200So, the monoclonal antibodies might interfere with nAChR activation by ACh. Monoclonal antibodies against these peptides specifically labelled a3-, a5-, and a7-subunits in all cultured neurons of the rat superior cervical ganglion, while only about half of the neurons revealed an a4-specific staining [5]. Interestingly, a completely different distribution of antibodies was found in rat intracardiac neurons, which suggests that nAChR subtypes expressed in these neurons are different from those in sympathetic ganglion neurons [5].In electrophysiological experiments, addition of a3-, a4-, and a7-specific monoclonal antibodies to the perfusing solution markedly reduced membrane currents evoked in non-dissociated neurons of the rat SCG by iontophoretically applied ACh (ACh current) and recorded with a whole-cell patch-clamp technique; the re...